【作者】 孙晓琳；

【导师】 周延民；

【作者基本信息】 吉林大学 ， 口腔临床医学， 2020， 博士

【摘要】 牙周炎是一种由菌斑聚集引起的炎症性疾病,如不加以控制会造成牙龈附着丧失,牙周袋形成,牙槽骨吸收,最终导致牙齿松动,甚至脱落。目前临床治疗牙周炎主要是通过机械方法进行表面清创。然而,牙周袋的局部因素,如深而窄的牙周袋,根分叉往往难以彻底清除细菌,多数情况下需要配合抗生素减少致病菌及毒素,以提高治疗效果。但是,随着细菌耐药性的出现,抗生素的应用受到了限制。因此,寻找一种更为安全有效的同时实现抑菌杀菌的疗法是非常必要的。近年来,应用抗菌光动力疗法（antimicrobial photodynamic therapy,aPDT）治疗牙周炎,不仅抑制了菌斑生长,而且避免了耐药菌株的产生,具有易操作、抗菌谱广、效率高的优点。虽然商品化的光敏剂已得到广泛的应用,但是仍存在一些亟待解决的问题:（1）光敏剂表面极性导致其生物利用率低;（2）由于牙周袋结构复杂,同时受龈沟液,唾液冲刷等影响,病损区光敏剂的浓度无法得到保障;（3）aPDT治疗是一个必须有氧参与的过程,而牙周炎形成的牙周袋是一个低氧环境,这就造成光敏剂介导的aPDT治疗牙周炎的效果受到影响。综上所述,如何提高病损区光敏剂有效浓度,如何促使aPDT在低氧环境下效果不受影响,对提高aPDT治疗牙周炎具有重要意义。本研究设计合成磁靶向自供氧纳米粒子,旨在研究其增强抗菌光动力疗法对牙周菌斑生物膜的影响,改善牙周微环境,为临床牙周炎的治疗提供新思路。在第二章中,首先通过两亲性硅烷作为载体,负载磁性Fe₃O₄,光敏剂二氢卟吩e6（Chlorin e6,Ce6）及香豆素6（Coumarin 6,C6）,合成磁靶向多功能纳米粒子（Fe₃O₄-silane@Ce6/C6 NPs）。然后,对纳米粒子进行性能的表征,生物安全性的评价,抗菌性能的评价和实时监测功能与磁靶向功能的评价。结果证明:该纳米粒子具有良好的水溶性、化学稳定性和生物安全性;对血链球菌（Streptococcus sanguinis,S.sanguinis）、牙龈卟啉单胞菌（Porphyromonas gingivalis,P.gingivalis）、具核梭杆菌（Fusobacterium nucleatum,F.nucleatum）三种牙周相关细菌均具有较强的抗菌作用;同时具有实时监测功能和磁性靶向的能力。在第三章中,对Fe₃O₄-silane@Ce6/C6 NPs进行改良,通过氧化还原沉淀反应在其表面吸附MnO₂,成功合成磁靶向自供氧纳米粒子（Fe₃O₄-silane@Ce6/C6@MnO₂ NPs）。对纳米粒子进行理化性能的表征和实时监测功能与磁靶向性能的评价。结果证明:MnO₂成功吸附于纳米粒子表面,并且该纳米粒子具有较强的氧化性;MnO₂的吸附不影响纳米粒子的实时监测功能和磁性靶向功能。在第四章,对Fe₃O₄-silane@Ce6/C6@MnO₂ NPs体外抗菌性能进行评价。结果证明:Fe₃O₄-silane@Ce6/C6@MnO₂ NPs具有良好的生物安全性;Fe₃O₄-silane@Ce6/C6@MnO₂ NPs能够显著增强aPDT对戈登氏链球菌（Streptococcus gordonii,S.gordonii）,P.gingivalis,F.nucleatum三种牙周相关细菌的单菌种生物膜和多菌种生物膜的抗菌作用;Fe₃O₄-silane@Ce6/C6@MnO₂ NPs能够通过抑制FimA-Ⅱ型基因（Fim A-Ⅱ型）和FimA-Ⅳ型基因（Fim A-Ⅳ型）、精氨酸特异性半胱氨酸蛋白酶基因（RgpA,RgpB）、赖氨酸特异性半胱氨酸蛋白酶基因（Kgp）的表达,可以阻碍细菌定植,干预细菌与宿主之间的相互作用,增强aPDT效应,在治疗牙周炎方面具有巨大潜力。在第五章中,首先,建立大鼠牙周炎症模型,然后探究基于Fe₃O₄-silane@Ce6/C6@MnO₂ NPs的aPDT对大鼠牙周炎症模型的影响。结果显示Fe₃O₄-silane@Ce6/C6@MnO₂ NPs介导的aPDT能够显著减轻牙龈炎症;Fe₃O₄-silane@Ce6/C6@MnO₂ NPs介导的aPDT组三种炎症因子阳性细胞率显著降低;Fe₃O₄-silane@Ce6/C6@MnO₂ NPs介导的aPDT组IL-1β,IL-6两种促炎因子基因表达显著下降,IL-10和Arg-1两种抑炎因子基因表达显著增加;同时Fe₃O₄-silane@Ce6/C6@MnO₂ NPs注射60天后,大鼠重要脏器未见损伤及炎症反应。综上所述,本研究利用两亲性硅烷作为载体,负载磁性Fe₃O₄,光敏剂Ce6及C6,提高Ce6的亲水性以增加其生物利用率,同时使纳米粒子具有磁靶向性能和实时监测Ce6药物浓度性能,最后在纳米粒子表面吸附MnO₂,利用MnO₂与H₂O₂产生O₂的特性,改善牙周低氧微环境,能够有效增强aPDT对牙周菌斑生物膜的抗菌效果,有效治疗大鼠牙周炎症,且具有较好的生物安全性。这种新型纳米粒子的合成为临床牙周炎的治疗提供了新思路,为磁靶向自供氧纳米粒子的临床应用提供了理论基础和实验支撑。更多还原

【Abstract】 Periodontitis is an inflammatory disease caused by oral biofilms.The inflammation leads to attachment loss,periodontal pocket formation,bone destruction,and ultimately,possible tooth loss.Traditionally,periodontal treatment has involved mechanical debridement to remove the biofilm and calculus that adhered to the infected root surfaces.But mechanical debridement fails to remove periodontal infection in a subset of cases without the ability to approach deep pockets and furcation.Adjunctive local antibiotics are often administered to eradicate or reduce the number of pathogenic bacteria and virulence factor.However,there is increasing attention about the abuse of antibiotics in periodontitis treatment due to the world’s public health concern for bacterial resistance.Therefore,new alternative strategies are needed to control biofilm and treat periodontal diseases.Antimicrobial photodynamic therapy（aPDT）is a promising antibacterial therapeutic for periodontitis,that advoid the emergence of drug-resistant strains,with the advantages of easy operation,broad-spectrum antibacterial activity and high efficiency.Although commercial photosensitizer has been widely used in clinic,there are still some problems to be solved urgently.The problems are as follow:（1）the hydrophobic nature of the most photosensitizer molecules leads to the reduced penetration of the photosensitizer into the biofilm matrix;（2）the traditional nanoparticles carrying photosensitizers agents is the low delivery efficiency due to the drainage of gingival crevicular fluid and high saliva fluid turnover;（3）continuous oxygen consumption in intrinsic hypoxic microenvironment would significantly hamper the efficacy of aPDT.In summary,how to improve the effective concentration of photosensitizer in the lesion area and how to make sure the efficacy of aPDT in intrinsic hypoxic microenvironment is of great significance to enhance the periodontitis treatment via aPDT.In the present study,magnetic-targeting and oxygen-generating nanoparticles were designed.The purpose of this study is to investigate the inhibitory effects of the nanoparticles via enhanced antibacterial photodynamic therapy（aPDT）against periodontal biofim.In Chapter two,firstly,magnetic Fe₃O₄,Chlorin e6（Chlorin e6,Ce6）and Coumarin 6（C6）were co-loaded with amphiphilic silane as the carrier to form multifunctional nanoparticles（denoted“Fe₃O₄-silane@Ce6/C6 NPs”）.Then,the physical and chemical properties of nanoparticles were characterized.Antimicrobial photodynamic therapy via Fe₃O₄-silane@Ce6/C6 NPs against periodontal bacteria were tested.Real-time monitor and the magnetic targeted antibacterial effects were also investigated.The results indicated the Fe₃O₄-silane@Ce6/C6 nanoparticles exhibited good water-solubility,chemical stability and biocompatibility.The multifunctional nanoparticles exerted strong anti-biofilm activity against Streptococcus sanguinis,Porphyromonas gingivalis,Fusobacterium nucleatum,with real-time monitoring and magnetically-targeting capacities.In Chapter three,the magnetic-targeting and oxygen-generating nanoparticles（Fe₃O₄-silane@Ce6/C6@MnO₂ NPs）were successfully synthesized by adsorption of MnO₂ on the surface of Fe₃O₄-silane@Ce6/C6 NPs with oxidation-reduction reaction.The physical and chemical properties of nanoparticles were characterized.Real-time monitor and the magnetic targeted antibacterial effects were also investigated.The results indicated the existence of MnO₂ and the strong oxidation susceptibility of the nanoparticles.The results confirmed the loaded MnO₂ did not affect the real-time monitoring function and magnetic targeting function of nanoparticles.In Chapter four,antimicrobial photodynamic therapy via magnetic-targeting and oxygen-generating nanoparticles against periodontal biofilm were tested.The Fe₃O₄-silane@Ce6/C6@MnO₂ NPs exhibited good biocompatibility.The results displayed strong anti-biofilm activity against single species biofilm and the multi-species biofilm of Streptococcus gordonii,Porphyromonas gingivalis,Fusobacterium nucleatum,and showed the suppression of the Fim A-Ⅱ,Fim A-Ⅳ,RgpA,RgpB and Kgp DNA expression,that can inhibit bacterial colonization and interfere with the interaction between bacteria and host.In Chapter five,firstly,established a model of periodontal inflammation in rats.Then,magnetic-targeting and oxygen-generating nanoparticles enhanced antimicrobial photodynamic therapy against periodontal inflammation in rats were investigated.The results showed that Fe₃O₄-silane@Ce6/C6@MnO₂ NPs-mediated aPDT could significantly reduce gingival inflammation.In addition,Fe₃O₄-silane@Ce6/C6@MnO₂ NPs-mediated aPDT group significantly reduced the positive cell rate of IL-1β,IL-6,TNF-α.Moreover,the results showed the increased IL-10 and Arg-1 with decreased IL-1b and IL-6.After the administration of Fe₃O₄-silane@Ce6/C6@MnO₂ NPs 60 days,there was no morphological changes or signs of inflammation occurred in the main organs.In conclusion,the present study developed co-loading Ce6 and C6 into the hydrophobic interspace between the octadecyl groups of oleic acid on the surface of Fe₃O₄ and the alkyl chains of silane,then loaded MnO₂ on the surface of Fe₃O₄-silane@Ce6/C6 NPs with oxidation-reduction reaction.The magnetic-targeting and oxygen-generating nanoparticles exerted excellent biocompatibility,real-time monitoring,and magnetically-targeting capacities.Meanwhile,the MnO₂coating modulates the hypoxic microenvironment by in situ generating O₂ through the reaction with endogenous H₂O₂,enhanced the aPDT against the periodontal biofilm to the rats periodontal inflammation treatment.The magnetic-targeting and oxygen-generating nanoparticles have great potential in antibacterial applications to inhibit the occurrence and progression of periodontitis.This novel design provides theoretical basis and experimental support using magnetic-targeting and oxygen-generating nanoparticles to administer aPDT.更多还原